RESEARCH ARTICLE 2477
Development 138, 2477-2485 (2011) doi:10.1242/dev.061770
© 2011. Published by The Company of Biologists Ltd
Drosophila Smt3 negatively regulates JNK signaling through
sequestering Hipk in the nucleus
Hai Huang1,2, Guiping Du1,2, Hanqing Chen1,2, Xuehong Liang1, Changqing Li1, Nannan Zhu1,2, Lei Xue3,
Jun Ma4 and Renjie Jiao1,*
SUMMARY
Post-translational modification by the small ubiquitin-related modifier (SUMO) is important for a variety of cellular and
developmental processes. However, the precise mechanism(s) that connects sumoylation to specific developmental signaling
pathways remains relatively less clear. Here, we show that Smt3 knockdown in Drosophila wing discs causes phenotypes
resembling JNK gain of function, including ectopic apoptosis and apoptosis-induced compensatory growth. Smt3 depletion leads
to an increased expression of JNK target genes Mmp1 and puckered. We show that, although knockdown of the homeodomaininteracting protein kinase (Hipk) suppresses Smt3 depletion-induced activation of JNK, Hipk overexpression synergistically
enhances this type of JNK activation. We further demonstrate that Hipk is sumolylated in vivo, and its nuclear localization is
dependent on the sumoylation pathway. Our results thus establish a mechanistic connection between the sumoylation pathway
and the JNK pathway through the action of Hipk. We propose that the sumoylation-controlled balance between cytoplasmic and
nuclear Hipk plays a crucial role in regulating JNK signaling.
INTRODUCTION
Small ubiquitin-related modifier (SUMO) is a polypeptide that is
covalently, but reversibly, conjugated to substrate proteins. This
post-translational modification, termed sumoylation, plays
important physiological roles by regulating various cellular
activities. Extensive studies have revealed that sumoylation plays
important roles in a variety of cellular processes such as
transcriptional regulation, nuclear-cytoplasmic transportation,
nuclear organization and DNA repair (Chen and Qi, 2010; Dou et
al., 2010; Heun, 2007; Lin et al., 2003; Rui et al., 2002). Similar to
the ubiquitylation process, sumoylation is achieved through
sequential enzymatic reactions. It is initiated by an E1 activating
enzyme (SAE1/SAE2, SUMO1-activating enzyme) that activates
the SUMO molecule at its C terminus, which is subsequently
linked to the E2-conjugating enzyme Ubc9 (ubiquitin-conjugating
enzyme 9), followed by E3 ligase-mediated transfer to a specific
substrate protein (Geiss-Friedlander and Melchior, 2007).
The gene that encodes SUMO was initially identified in
Saccharomyces cerevisiae (Meluh and Koshland, 1995). In
mammalian cells, there are three SUMO genes, whereas only a
single gene, smt3, exists in Drosophila (Huang et al., 1998;
Johnson et al., 1997; Su and Li, 2002), making Drosophila a useful
experimental system in which to study the biological functions of
sumoylation. Several studies in Drosophila have suggested
1
State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, the
Chinese Academy of Sciences, Datun Road 15, Beijing 100101, China. 2Graduate
School of the Chinese Academy of Sciences, Beijing 100080, China. 3Shanghai Key
Laboratory for Signaling and Diseases, School of Life Science and Technology, Tongji
University, 1239 Siping Road, Shanghai 200092, China. 4Divisions of Biomedical
Informatics and Developmental Biology, Cincinnati Children’s Research Foundation,
3333 Burnet Avenue, Cincinnati, OH 45229, USA.
*Author for correspondence ([email protected])
Accepted 22 March 2011
different functions of sumoylation, including in the regulation of
cell signaling during development and ecdysteroid biosynthesis, but
their underlying mechanisms remain largely unclear (Miles et al.,
2008; Nie et al., 2009; Talamillo et al., 2008).
The c-Jun N-terminal kinase (JNK) signaling is an evolutionarily
conserved pathway, which is activated in response to
environmental stress, apoptotic signals and proinflammatory
cytokine tumor necrosis factor (TNF) (Liu et al., 1996; Moreno et
al., 2002; Ryoo et al., 2004; Xia et al., 1995). In Drosophila, JNK
is encoded by the gene basket (bsk). The upstream regulators of
Bsk include a series of kinases that form a signaling cascade
(Stronach and Perrimon, 2002; Takatsu et al., 2000; Tateno et al.,
2000; Xue et al., 2007). MSN, a MAPK kinase kinase kinase
(MAPKKKK) receives signals from cell surface receptors and
initiates this signaling cascade (Liu et al., 1999; Xue et al., 2007).
While the main signaling pathway transmits from MSN to JNK
hierarchically, other factors connected to this main pathway also
function in fine-tuning of the signaling, especially in activating or
repressing the JNK activity (Chen et al., 2002; Neisch et al., 2010;
Shanley et al., 2001; Yang et al., 1997).
One of the factors suggested to have a role in activating JNK is
the homeodomain-interacting protein kinases (Hipks) (Hofmann et
al., 2003; Lan et al., 2007; Li et al., 2005). Hipks are a family of
serine/threonine kinases that are initially identified as the regulators
of transcriptional co-repressors (Choi et al., 2005; Kim et al., 1998;
Sung et al., 2005; Zhang et al., 2003). Although there are four
members of Hipk proteins in vertebrates, Drosophila has only one
ortholog: Hipk. The Drosophila Hipk shares the highest homology
with mammalian Hipk2 (Choi et al., 2005; Link et al., 2007). Hipk
functions in a variety of biological processes, some of which are in
common with the JNK pathway, such as apoptosis and
morphogenesis (Inoue et al., 2010; Isono et al., 2006; Link et al.,
2007; McEwen et al., 2000; Zhang et al., 2003). However, an
operational connection between Hipk and JNK at a mechanistic
level has not been well established.
DEVELOPMENT
KEY WORDS: Drosophila, Smt3, JNK, Hipk, Sumoylation
2478 RESEARCH ARTICLE
MATERIALS AND METHODS
Drosophila strains
Flies were reared on a cornmeal and agar medium at 25°C according to
standard protocols. The RNAi lines of smt3 described previously
(Talamillo et al., 2008) were kindly provided by Dr Rosa Barrio (CIC
bioGUNE, Bizkaia, Spain). The smt3 mutant allele referred to as sumo04493
in this study, which harbors a P-element insertion in the upstream of the
transcriptional start site (5⬘-UTR) of smt3 gene that impairs the
transcription of smt3 was obtained from the Bloomington Stock Center.
The hipk-RNAi allele and UAS-hipk have been described previously (Lee
et al., 2009a; Lee et al., 2009b). The UAS-smt3 flies have been described
previously (Nie et al., 2009; Takanaka and Courey, 2005). The RNAi
stocks of smt3 and hipk are available from the Vienna Drosophila RNAi
Center (VDRC) and Fly Stocks of National Institute of Genetics (NIGFLY).
Immunohistochemistry and microscopy
Wandering third instar larvae with correct genotypes were collected and
dissected in cold phosphate-buffered saline (PBS). Imaginal discs were
fixed in 4% paraformaldehyde. After proper washes, the discs were
blocked in 10% goat serum, and stained with different primary antibodies
(see below). Subsequently, corresponding fluorescent secondary antibodies
(1:100, Jackson ImmunoResearch) were used for signal detection. The
images were photographed with Leica confocal microscope SP5. The
primary antibodies and their dilutions used for immunohistochemistry are
as follows: antibodies against cleaved Caspase 3 (1:100) (Cell Signaling),
Wingless antibodies (1:100) (Developmental Studies Hybridoma Bank,
DSHB), mouse anti-Mmp1 (1:50) (DSHB 3A6B4/5H7B11/3B8D12) and
anti-HA antibodies (1:100) (Roche).
Western blot and immunoprecipitation
The extracts were prepared as previously described (Huang et al., 2010).
Adult heads were cut from newly enclosed flies and homogenized in
radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl (pH 8.0),
150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF] in
the presence of a protease inhibitor cocktail. After incubation on ice for 15
minutes, the lysates were spun down at a maximum speed. The
supernatants were used either for immunoblot or for coimmunoprecipitation assays.
The samples were mixed with 2⫻SDS buffer [125 mM Tris-HCl (pH
6.8), 20% glycerol, 2% SDS, 0.1% Bromophenol Blue, 20% 2mercaptoethanol], boiled for 5 minutes and centrifuged at the maximum
speed at room temperature for 5 minutes. The supernatants were then
applied to SDS-polyacrylamide gel and transferred to a PVDF membrane.
For western blotting, the membranes were blocked for 1 hour at room
temperature and probed with anti-HA antibody (Roche), anti-SUMO
antibody (Abgent) and anti-Actin antibody (Santa Cruz), followed by
horseradish-peroxidase linked secondary antibody. The signals were
detected using SuperSignal West Pico Trial Kit (Thermo Scientific).
For co-immunoprecipitations, antibodies as well as control IgG, were
coupled to Dynabeads Protein A/G (Invitrogen). The extracts were
incubated with the beads for 6 hours at 4°C and eluted with SDS-loading
buffer [125 mM Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, 0.05%
Bromophenol Blue, 10% 2-mercaptoethanol] before SDS-PAGE for
immunoblotting.
Fractionation assay
Fly heads from newly enclosed flies were collected. The nuclear and
cytoplasmic fractions were separated by the NER-PER Nuclear and
Cytoplasmic Extraction Reagents (Thermo Scientific) following the
manufacturer’s instructions. The samples were mixed with 2⫻SDS buffer
[125 mM Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, 0.1% bromophenol
blue, 20% 2-mercaptoethanol], boiled and applied for sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before western
analysis.
TUNEL assay
The wing imaginal discs of proper genotypes were dissected in ice-cold
PBS and fixed in 4% formaldehyde before being permeabilized in 1%
Triton X-100 for 30 minutes. After sufficient washes, samples were
incubated in the mixture of Enzyme and Label solutions (Beyotime Kit) at
37°C for 1.5 hours. The rest of the experiment was carried out by following
the manufacturer’s instructions.
RNA interference and immunostaining of cultured S2 cells
S2 cells were maintained in Schneider’s insect medium with 10% fetal
bovine serum and antibiotics at 25°C, following the standard protocol.
DNA template for RNA production was amplified with primers containing
T7 promoter. The pair of primers for Smt3 is 5⬘TAATACGACTCACTATAGGGGGCGTGTAGCTGTAGCAGAAGC-3⬘
and 5⬘-CCCTATAGTGAGTCGTATTACTTATGGAGCGCCACCAGTCTG-3⬘. The primers for GFP are 5⬘-TAATACGACTCACTATAGGGAGATCTATGGTGAGCAAGGG-3⬘ and 5⬘-CCCTATAGTGAGTCGTATTACTTGTACAGCTCGTCCATGC-3⬘. The DNA templates
were in vitro transcribed into dsRNAs using the RiboMAX Large Scale
RNA production System-T7 (Promega). S2 cells were seeded on
polylysine-treated coverslips in dishes. dsRNA was introduced into
cultured S2 cells, using standard calcium phosphate transfection method 3
days before immunostaining. pAc5.1A-HA-Hipk expression plasmids were
introduced into the S2 cell 36 hours prior to immunostaining. The
transfected cells were fixed in 4% paraformaldehyde. After primary
antibodies and fluorescent secondary antibodies incubation, the images
were obtained with a Leica confocal microscope SP5.
RESULTS
Drosophila Smt3 is essential for development and
tissue growth
In Drosophila, smt3 encodes the SUMO molecule that is
ubiquitously expressed and predominantly distributed in the
nucleus (Lehembre et al., 2000; Nie et al., 2009; Talamillo et al.,
2008). A recent proteomic study has identified over 100 Drosophila
proteins as substrates of sumoylation, proteins that play important
roles in early embryonic development (Nie et al., 2009). Talamillo
and colleagues reported that Smt3 knockdown produces
developmental arrest and alters the ecdysteroid synthesis that is
essential for metamorphosis (Talamillo et al., 2008). To further
investigate the biological functions of Smt3 during development,
we analyzed a mutant allele of smt3, smt304493, which harbors a Pelement insertion in the upstream of the transcription start site (Nie
et al., 2009). The mutant animals fail to survive beyond the second
instar larval stage, and ubiquitous knockdown of smt3 causes
developmental arrest at the pupal stage (data now shown). These
results are consistent with those described recently (Talamillo et al.,
2008) and further demonstrate that Smt3 is essential for
development. To gain a better understanding of the functional role
of Smt3 during development, we used Gal4 lines to deplete Smt3
in a tissue-specific manner. Our results show that either ey-Gal4driven or A9-Gal4-driven expression of an smt3-RNAi construct
DEVELOPMENT
In this study, we show that knockdown of the SUMO gene
(smt3) leads to an upregulation of the JNK signaling pathway in
Drosophila. In a genetic screen for suppressors of Smt3 depletioninduced phenotype in the wing, we identified Hipk. We show that
Hipk knockdown suppresses Smt3 depletion-induced JNK
signaling upregulation. We further show that Drosophila Hipk is a
target of sumoylation and its proper nuclear localization is
dependent on the sumoylation pathway. Our results suggest a
model, in which the sumoylation pathway normally keeps Hipk
inside the nucleus; but downregulation of this pathway causes a
translocation of Hipk to the cytoplasm, leading to an activation of
JNK signaling. Our study thus provides a mechanistic connection
between the subcellular localization of Hipk, a process regulated
by sumoylation and JNK signaling.
Development 138 (12)
Fig. 1. Smt3 knockdown flies display developmental defects in
various contexts. (A-A⬘) Eye development is compromised upon Smt3
knockdown specifically in the eyes [compare A (ey-Gal4/+) with A⬘
(ey>smt3-IR)]. (B-B⬘) Wing development is defective when Smt3 is
depleted under the control of A9-Gal4. Adult wings of A9-Gal4/+ (B)
and A9>smt3-IR (B⬘) flies are shown. (C-C⬘) Light microscopy images
showing adult thoraxes of control (C, pnr-Gal4/+) and Smt3
knockdown flies (C’, pnr>smt3-IR). Note the midline defects in the
notum and missing scutellum for the Smt3 knockdown flies.
severely reduced the sizes of the eye or the wing, respectively (Fig.
1A⬘,B⬘; see Fig. 1A,B for wild-type controls). The specificity of
the smt3-RNAi construct was validated by a genetic rescue
experiment with UAS-smt3 transgene (see Fig. S1 in the
supplementary material). In addition, depletion of Smt3 in the
notum and scutellum under the control of pnr-Gal4 caused a defect
in the midline of the notum and a loss of scutellum (Fig. 1C⬘; see
Fig. 1C for wild-type control). These results demonstrate a crucial
role of Drosophila smt3 in development, and its tissue-specific
disruption leads to corresponding tissue losses.
Knockdown of smt3 leads to apoptosis and
activates wg expression in the wing discs
The tissue loss caused by Smt3 depletion can be attributed to
several events, including cell apoptosis. To test whether apoptosis
is induced upon Smt3 depletion, we stained for the cleaved Caspase
3 that marks cells undergoing apoptosis. en-Gal4 was used to
specifically knockdown smt3 in the posterior compartment of the
wing disc. As shown in Fig. 2A, the GFP signals mark the territory
where en-Gal4 is expressed. When compared with the anterior
compartment where relatively few apoptotic cells were observed,
the GFP-positive posterior region exhibited a significantly
increased population of Caspase 3-positive cells. As shown in Fig.
S2 in the supplementary material, the apoptotic cells were also
detected in the TUNEL assay. Together, these results suggest that
Smt3 depletion promotes apoptosis.
The imaginal discs that develop into adult appendages can
recover from damages caused by physical injury or apoptosis
through regenerative growth (McEwen and Peifer, 2005; Smith-
RESEARCH ARTICLE 2479
Fig. 2. RNAi depletion of Smt3 induces apoptosis and ectopic Wg
expression. (A)Immunostaining images showing apoptotic cells
detected by Caspase 3 signals (middle panels) in wing discs. Left panels
mark posterior wing compartments with en-Gal4 driven GFP
expression. (B)Wingless expression pattern in wild-type (en-Gal4/+,
upper panels) and Smt3 knockdown (en>smt3-IR, lower panels) wing
imaginal discs. Scale bars: 75m.
Bolton et al., 2009; Wang et al., 2009). The expression of the
Wingless (Wg) morphogen in surviving cells is required for this
regenerative repair (Ryoo et al., 2004; Smith-Bolton et al., 2009).
We sought to determine whether the Smt3 depletion-induced
apoptosis may trigger regenerative growth by examining wg
expression. In the control discs, Wg forms a stripe at the dorsalventral boundary (Fig. 2B, upper panels). Upon Smt3 depletion
under the control of en-Gal4 in the posterior region, the Wg
morphogen expression became obscure at the D/V boundary (Fig.
2B, lower panels). In addition, ectopic expression of Wg was
induced in the surviving cells of the entire posterior wing pouch,
suggesting that regenerative growth takes place in this part of the
disc (Fig. 2B, lower panels). Taken together, our results suggest
that depletion of Smt3 causes apoptosis and induces Wg
morphogen ectopic expression.
Reduction of Smt3 promotes JNK signaling
activity
Both apoptosis and apoptosis-induced compensatory proliferation
are governed by the JNK signaling pathway (Igaki et al., 2002;
Moreno et al., 2002; Perez-Garijo et al., 2009). To determine
whether JNK is required for Smt3 depletion-induced phenotypes,
we blocked JNK activity simultaneously in the Smt3 knockdown
DEVELOPMENT
Smt3 suppresses JNK signaling
2480 RESEARCH ARTICLE
Development 138 (12)
Fig. 3. The Smt3 depletion-induced phenotype is dependent on
Drosophila JNK. (A)Blocking JNK signaling rescues wing growth
defects in A9>smt3-IR flies. Genotypes: upper panels are A9-Gal4/+;
UAS-bskDN/+ and A9-Gal4/+; UAS-bsk-IR/+. Middle panel is A9>smt3-IR
fly (A9-Gal4/+; UAS-smt3-IR/+). Bottom panels are A9-Gal4/+; UASsmt3-IR/UAS-bskDN and A9-Gal4/+; UAS-smt3-IR/UAS-bsk-IR.
(B)Downregulation of JNK signaling rescues the apoptosis resulting
from Smt3 depletion in en>smt3-IR. Wing discs were immunostained
with Caspase 3 antibody (middle panels). (C)Downregulation of JNK
signaling suppresses ectopic Wg expression and restores the D/V
boundary Wg pattern in en>smt3-IR animals. Red shows Wg signal.
Scale bar: 75m.
tissues through the use of RNAi against the Drosophila JNK (bsk)
or the use of a dominant-negative form of Bsk (Fig. 3A-C). Three
lines of evidence show that both apoptosis and ectopic wg
expression induced by Smt3 knockdown are dependent on JNK
activity. First, either depletion of Drosophila JNK by bsk-RNAi or
expression of a dominant-negative form bskDN rescued the small
wing phenotype induced by Smt3 knockdown (Fig. 3A). Second,
JNK abrogation substantially reduced the apoptosis in the
en>smt3-IR discs as shown in Fig. 3B. Finally, the ectopic
expression of wg no longer occurs in the Smt3 knockdown area
when JNK is inactivated through bsk-RNAi; instead, these
experimental discs exhibit a wg expression pattern similar to that
of wild-type control (Fig. 3C). These observations demonstrate that
JNK activity is required to manifest the effects of Smt3 depletion
in establishing the observed wing phenotypes.
To monitor the JNK pathway activity directly, we analyzed two
reporters for their dependence on Smt3. The first reporter is puclacZ (pucE69). puckered (puc) is a transcriptional target of JNK and
is activated in the proximal peripodial cells of the wild-type wing
discs (Agnes et al., 1999; McEwen et al., 2000; Miotto et al., 2006;
Zeitlinger and Bohmann, 1999). The second reporter is the Matrix
metalloproteinase 1 (Mmp1) gene, another downstream
transcriptional target of JNK (Rodahl et al., 2009; Uhlirova and
Bohmann, 2006). Our results show that Smt3 depletion, under the
control of en-Gal4 and marked by the GFP-positive cells, led to
both ectopic puc-lacZ reporter activity (Fig. 4, pucE69) and
increased Mmp1 signals (Fig. 4, Mmp1), when compared with the
DEVELOPMENT
Fig. 4. Depletion of Smt3 upregulates JNK signaling.
Immunofluorescent images show localization of -galactosidase in en/+
and en>smt3-IR larval wing discs of heterozygous pucE69 flies. Wing
discs of en/+ and en>smt3-IR larvae were immunostained with Mmp1
antibody to indicate the activity of the JNK pathway in the posterior
region. Scale bars: 75m.
Smt3 suppresses JNK signaling
RESEARCH ARTICLE 2481
GFP-negative and Smt3-positive cells in the anterior region. Taken
together, these results provide direct evidence that Smt3 depletion
promotes the JNK activity in vivo.
Hipk is required for JNK signaling activation
induced by Smt3 depletion
The JNK signaling pathway is regulated by multiple factors
(Huang et al., 2009; Minden and Karin, 1997). To identify possible
effectors through which Smt3 regulates JNK activity, we took
advantage of the small-wing phenotype in A9>smt3-IR flies as a
tool to screen for genetic suppressor(s). We identified the
homeodomain interacting protein kinase (Hipk) as one such
suppressor. Fig. 5A shows that knockdown of Hipk was sufficient
to rescue the wing developmental defects of A9>smt3-IR flies.
Both Smt3-depletion-induced apoptosis (monitored by Caspase 3
cleavage) and transcriptional activation of Mmp1 were attenuated
by the Hipk depletion (Fig. 5B,C). Genetic experiments suggest
that Hipk is required for full activation of JNK (see Fig. S3 in the
supplementary material). In addition, pnr-Gal4-driven
overexpression of Hipk caused thorax and scutellum defects that
resemble the JNK gain-of-function phenotype and Smt3-depletion
phenotype (see Fig. S4 in the supplementary material). These
results suggest that Hipk plays an important role in the JNK
activation induced by Smt3 depletion.
To determine whether overexpression of Hipk alone is
sufficient to induce JNK pathway activation, we artificially
expressed Hipk under the control of en-Gal4 in the posterior
region of the wing discs. Our results show that overexpression
of Hipk resulted in only a slight increase of Caspase 3-positive
cells and a mild elevation of Mmp1 expression (Fig. 6),
indicating that Hipk overexpression alone activates JNK pathway
only weakly, and is insufficient to induce strong JNK signaling.
To further investigate the relationship between Hipk action and
the Smt3 status for their roles in JNK activation, we used the
sensitive eye phenotype for our investigation. As shown
previously, the severity of rough eyes reflects the strength of
JNK-dependent apoptosis (Igaki et al., 2002; Moreno et al.,
2002; Tiwari and Roy, 2009; Xue et al., 2007). Here, we
simultaneously expressed UAS-hipk and smt3-RNAi constructs
under the control of GMR-Gal4. Our results show that, when
compared with the wild-type ommatidia (Fig. 7A), Smt3
knockdown driven by GMR-Gal4 caused a slight irregular
ommatidia pattern (Fig. 7B; see Fig. S5 in the supplementary
material). GMR-Gal4-driven expression of Hipk did not lead to
any obvious abnormality (Fig. 7C), which is consistent with the
observation that overexpression of Hipk alone activates only
mild apoptosis (Fig. 6). However, simultaneous expression of
Hipk and smt3 knockdown led to significantly enhanced rough
eyes with reduced size and fused ommatidia (Fig. 7D; see Fig.
S5 in the supplementary material). At the molecular level,
overexpression of Hipk synergistically promoted the expression
of JNK target gene Mmp1 and ectopic apoptosis caused by Smt3
knockdown (see Fig. S6A,B in the supplementary material).
These results demonstrate a synergy between Hipk
overexpression and Smt3 removal in JNK activation, suggesting
that the role of Hipk on JNK is dependent on the status of the
DEVELOPMENT
Fig. 5. The JNK signaling
activation induced by Smt3
depletion depends on the action
of Hipk. (A)Knockdown of Hipk
rescues the wing growth defects in
A9>smt3-IR flies. Genotypes: upper
panel is A9-Gal4/+; UAS-hipk-IR/+.
Middle panel is A9-Gal4/+; UASsmt3-IR/+. Bottom panel is A9Gal4/+; UAS-smt3-IR/UAS-hipk-IR.
(B)Apoptosis induced by Smt3
depletion is reversed when Hipk is
knocked down. Apoptotic cells were
visualized using Caspase 3 antibody
(middle panels). (C)RNAi ablation of
Hipk offsets the upregulation of JNK
activity (as indicated by the Mmp1
signals) induced by Smt3 knockdown.
Wing discs were stained with Mmp1
antibody (middle panels). Scale bars:
75m.
Fig. 6. Overexpression of Hipk triggers a mild increase of JNK
signaling. Detectable apoptosis indicated by Caspase 3 signals (upper
panels, arrowheads) and slight Mmp1 upregulation (lower panels) in
the posterior region of the wing discs were induced by overexpression
of Hipk (en>hipk). Scale bars: 75m.
sumoylation pathway. We will further discuss the implications of
these findings regarding the operational relationship between
Hipk and Smt3 on JNK activation (see below and Discussion).
Hipk is sumoylated in vivo in the presence of
Smt3
As Hipk regulates JNK signaling in a manner that is dependent on
sumoylation perturbation (i.e. Smt3 depletion), it is possible that
Hipk may itself be a target of sumoylation. To test this possibility
directly, we performed an immunoprecipitation assay using protein
extracts from the heads of the flies that express HA-tagged Hipk
transgene under the control of GMR-Gal4 (GMR>Hipk-HA). In our
analysis, anti-HA antibody was used for immunoprecipitation,
followed by immunoblot using an anti-SUMO antibody. A HipkHA band was detected in this experiment by the anti-SUMO
antibody (Fig. 8A, lane 2). Importantly, the amount of this band is
reduced by Smt3 knockdown (Fig. 8A, lane 3). In a reciprocal
experiment, Hipk was detected in the products immunoprecipitated
by the anti-SUMO antibody (Fig. 8B). Together, these results
provide evidence that Hipk is sumoylated in vivo.
Depletion of Smt3 leads to translocation of Hipk
from the nucleus to the cytoplasm
Our experiments described thus far suggest that sumoylation
suppresses the ability of Hipk to activate the JNK signaling
pathway (Fig. 5A-C). They also show that Hipk itself is
sumoylated (Fig. 8), suggesting that the sumoylation status of Hipk
may be crucial for its role in regulating JNK activation. It has been
reported that sumoylation can alter protein localization and/or
change protein conformation (Geiss-Friedlander and Melchior,
2007; Girdwood et al., 2004; Lin et al., 2003; Sanchez et al., 2010;
Zhong et al., 2000). To investigate the role of sumoylation on Hipk
subcellular localization, we separated the nuclear and cytosolic
fractions of the extracts from Hipk-expressing adult fly heads with
or without smt3-RNAi expression. Western blot with anti-SUMO
antibody detected sumoylated Hipk primarily in the nuclear
fraction (Fig. 9A, SUMO). Although Smt3 knockdown did not
alter the total amount of Hipk (Fig. 8A, Hipk-HA, compare lanes
2 and 3), its nuclear abundance was decreased upon the reduction
Development 138 (12)
Fig. 7. Overexpression of Hipk and knockdown of smt3
synergistically induce developmental defects in the eye. Light
microscopic photographs showing Drosophila adult eyes. Genotypes:
(A) GMR-Gal4/+, (B) GMR-Gal4/+; UAS-smt3-IR/+, (C) GMR-Gal4/+;
UAS-hipk/+ and (D) GMR-Gal4/+; UAS-smt3-IR/UAS-hipk.
of Smt3 (Fig. 9A, nuclear fraction of Hipk-HA). Concomitantly,
the level of Hipk was increased in the cytosolic fraction (Fig. 9A,
cytosolic fraction of Hipk-HA). These results suggest a
translocation of Hipk from the nucleus to the cytoplasm when the
sumoylation pathway is compromised by Smt3 depletion.
To monitor directly the effects of sumoylation on the dynamic
localization of Hipk, we performed immunostaining experiments
using an HA antibody that detects an HA-tagged Hipk protein. Fig.
9B (upper panels) shows that Hipk is ubiquitously expressed with
a primary localization in the nucleus, a pattern resembling the
subcellular localization of Smt3 (Talamillo et al., 2008). The
nuclear accumulation of Hipk is in agreement with its reported
activity of interacting with other transcriptional regulators (Choi et
al., 2005; Zhang et al., 2003). To further evaluate the effect of
sumoylation on Hipk localization, we conducted immunostaining
of HA-tagged Hipk proteins on endogenous Drosophila tissues and
in vitro cultured Drosophila S2 cells. In the case of Smt3
knockdown, the amount of nuclear Hipk was significantly reduced
when compared with the wild-type control (Fig. 9B, Hipk-HA,
lower panels; also see Fig. 9A, nuclear fraction). Meanwhile, a
noticeable increase of the Hipk signals was detected in the
cytoplasmic fraction upon the perturbation of sumoylation pathway
by Smt3 depletion (Fig. 9B, Hipk-HA, lower panels; also see Fig.
9A, cytosolic fraction). The nucleus-to-cytoplasm translocation of
Hipk induced by the impairment of the sumoylation pathway was
further confirmed by experiments in Drosophila S2 cells (see Fig.
S7 in the supplementary material). Together, our fractionation and
immunostaining results suggest a role of the sumoylation pathway
in regulating the subcellular localization of Hipk.
DISCUSSION
Sumoylation is a post-translational modification that regulates
multiple biological activities by modifying a variety of different
substrates. In this study, we show that tissue-specific perturbation
of the sumoylation pathway activates the JNK signaling pathway.
In particular, knockdown of the Drosophila SUMO gene smt3
recapitulates several key gain-of-function features of the JNK
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2482 RESEARCH ARTICLE
Smt3 suppresses JNK signaling
RESEARCH ARTICLE 2483
Fig. 8. Hipk is sumoylated in vivo. (A)Extracts were prepared from
the heads of adult wild-type (GMR-Gal4/+, lane 1), GMR>hipk-HA
(GMR-Gal4/+; UAS-hipk-HA/+, lane 2) and GMR>hipk-HA, smt3-IR
(GMR-Gal4/+; UAS-hipk-HA/UAS-smt3-IR, lane 3) flies. The proteins
were pulled down with anti-HA antibodies, separated by SDS-PAGE,
and immunoblotted with anti-SUMO and anti-HA antibodies
independently. (B)In the reverse experiment, immunoprecipitates were
collected with anti-SUMO antibody, and detected by antibodies against
HA. Control IgG serves as a negative control.
Fig. 9. Depletion of Smt3 translocates Hipk from the nucleus to
the cytoplasm. (A)Western blot analyses of the cytosolic and the
nuclear extracts from heads of the GMR>hipk-HA (GMR-Gal4/+; UAShipk-HA/+) and GMR>hipk-HA fly lines with compromised sumoylation
pathway (GMR-Gal4/+; UAS-hipk-HA/UAS-smt3-IR). Anti-HA antibody
is used to measure the nuclear and cytoplasmic abundance of Hipk.
Anti-SUMO antibody was used to detect sumoylated Hipk-HA pulled
down by anti-HA antibody. Histone H3 and Actin antibodies are used
for loading controls. The quantification of the western blot results is
shown below each lane. The ratios (numbers below the bands of Smt3IR samples) represent the relative intensity of the signals in the absence
of Smt3 (GMR-Gal4/+; UAS-hipk-HA/UAS-smt3-IR) compared with that
in the presence of Smt3 (GMR-Gal4/+; UAS-hipk-HA/+) that were
normalized to 1. (B)Wing imaginal discs of third instar larvae expressing
HA tagged Hipk (A9>hipk-HA) in the presence of Smt3 (upper panels)
and in the absence of Smt3 (lower panels) were stained with anti-HA
antibodies (green channels). Note the reduction of Hipk signals in the
nuclei when Smt3 is depleted. Nuclei were visualized using DAPI
staining (red channels). Scale bars: 4m.
example, Hipk1 and Hipk2 have functionally redundant roles in
mediating cell proliferation and apoptosis during development (Inoue
et al., 2010; Isono et al., 2006). Hipk1 interacts with transcription
factor c-Myb (Matre et al., 2009), while Hipk2 phosphorylates
transcriptional co-repressor Groucho (Choi et al., 2005), suggesting
their distinct roles in transcription regulation. Therefore, it would be
interesting to elucidate whether Drosophila Hipk executes the
functions of all the mammalian counterparts, although Drosophila
Hipk shares most homology with Hipk2. This all-in-one mode of
Hipk function requires different strategies to regulate its functions.
Previous studies have shown that Drosophila Hipk promotes various
signaling pathways such as the Wnt pathway through stabilizing
Armadillo, and the Notch pathway through inhibiting the global corepressor Groucho (Lee et al., 2009a; Lee et al., 2009b). In this work,
DEVELOPMENT
pathway, including apoptosis and wg ectopic expression. These
results suggest that sumoylation plays a crucial role in regulating
JNK signaling. Further experiments demonstrate that Hipk is
responsible for Smt3 depletion-induced JNK activation. Our
experiments show that Hipk itself is sumoylated (Fig. 8) and that
its nuclear localization is dependent on the sumoylation pathway
(Fig. 9). Based on these findings, we propose a model in which
Hipk is normally kept in the nucleus, but a compromised
sumoylation pathway (such as that produced by depletion of Smt3)
allows some Hipk molecules to translocate to the cytoplasm and
activate the JNK signaling pathway.
Sumoylation regulates the biological activities of its substrates
through several distinct mechanisms. These mechanisms include
altering subcellular localization of its substrate proteins and/or
molecular shuttling between the nucleus and the cytoplasm (Ishov
et al., 1999; Li et al., 2005), mediating protein-protein interactions
(Muller et al., 1998), locking its substrates in a particular
conformational state (i.e. active or inactive) (Girdwood et al., 2004)
or altering protein stability and clearance (Zhang et al., 2003). Our
study highlights the importance of sumoylation-dependent
subcellular localization of Hipk in regulating its biological
activities. We propose that sumoylation normally restricts Hipk to
the nucleus and facilitates the execution of its nuclear functions,
such as interaction with and phosphorylation of transcriptional corepressors (Choi et al., 2005; Ecsedy et al., 2003; Zhang et al.,
2003). However, unsumoylated or desumoylated Hipk becomes
accessible to the cytoplasm for executing its cytoplasmic
function(s). As shown in our study, one such cytoplasmic function
of Hipk is to modulate the JNK signaling pathway.
Hipk family members play roles in different biological processes,
such as cell cycle progression, p53-dependent apoptosis, and
transcriptional regulation (D’Orazi et al., 2002; Pierantoni et al.,
2001; Zhang et al., 2003). The mammalian cells contain four Hipk
proteins that perform overlapping, but distinct functions. For
we report for the first time that Drosophila Hipk potentiates JNK
signaling through a sumoylation-dependent regulation of its
subcellular localization. Our study and the work from Verheyen’s
laboratory underscore the roles of Drosophila Hipk both inside and
outside of the nucleus in fine-tuning signaling pathways. It remains
to be determined precisely how Hipk regulates the JNK pathway and
whether it involves a direct mechanism such as phosphorylating
relevant components of this pathway.
The subcellular localization of Hipk represents an important
mechanism in defining its functional specificity. In particular, Hipk
controls the degradation of transcriptional co-repressor CtBP inside
the nucleus (Zhang et al., 2003), while the cytoplasmic Hipk
interacts with the nonhistone chromosomal factor Hmga1 (highmobility group A1) to inhibit cell growth (Pierantoni et al., 2001).
Hipk has also been shown to, within the speckled subnuclear
structures, interact with p53 to promote its phosphorylation
(Gostissa et al., 2003; Moller et al., 2003). Our results presented in
this report show that Hipk is normally sequestered in the nucleus
but gains access to the cytoplasm, upon sumoylation perturbation,
to activate the JNK signaling pathway. The idea that the subcellular
localization of Hipk is crucial for its functional specificity also
explains why overexpressing Hipk alone did not result in a robust
activation of JNK (Fig. 6). We suggest that, without sumoylation
perturbation, the majority of transgene-expressed Hipk is, like the
endogenously expressed Hipk, sumoylated and kept in the nucleus,
making it inaccessible to activating JNK.
The JNK signaling pathway is composed of stepwise actions of
kinases (Geuking et al., 2005; Geuking et al., 2009). The canonical
JNK pathway receives signals from death stimuli, such as tumor
necrosis factor (TNF) and oxidative stresses. In addition to the JNK
pathway, other factors such as Hipk proteins are also stimulated by
a variety of stresses. For example, the human HIPK1 responds to
the stimulation of TNF to relocate itself from the nucleus to the
cytoplasm (Li et al., 2005). In addition, the mammalian Hipk2
phosphorylates p53 in response to UV irradiation (D’Orazi et al.,
2002; Hofmann et al., 2002; Zhang et al., 2003) and phosphorylates
cyclic AMP response element-binding protein (CREB) to cope
with genotoxic stress (Sakamoto et al., 2010). We propose that
stress signals such as TNF may activate not only the canonical JNK
pathway but also the Hipk-dependent JNK activation
mechanism(s). The idea that Hipk acts downstream of TNF is
consistent with our genetic evidence that RNAi ablation of Hipk
partially rescues the Drosophila TNF (Egr)-induced phenotype in
the eye (see Fig. S3A in the supplementary material). A major
finding of our current study is that it establishes a cross-regulation
between the sumoylation and the JNK pathways through the action
of Hipk. We currently do not know whether TNF or even JNK
itself may regulate the sumoylation pathway, but it remains an
interesting possibility that will require further investigation. We
note that the relationship between sumoylation and JNK pathways
is likely to be more complex than Hipk-mediated action described
in our current work. It has been shown that sumoylation is required
for Axin-mediated JNK activation (Rui et al., 2002). Thus, it is
possible that sumoylation may have different, even opposite, effects
on the JNK pathway through distinct sumoylation targets. The
robust increase of the JNK activity detected in Smt3-depleted cells
in our study demonstrates an overall negative role of the
sumoylation pathway in JNK signaling.
Acknowledgements
We thank Dr Rosa Barrio, Dr Esther Verheyen, Dr Albert Courey, Dr Xun
Huang, the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi
Center and Fly Stocks of National Institute of Genetics (NIG-FLY) for fly stocks;
Development 138 (12)
the Developmental Studies Hybridoma Bank for antibodies; and Haidong
Huang for technical assistance and suggestions. This work has been financially
supported by the 973 program (2009CB918702) and the NSFC (31071087,
30623005 and 30771217). We are grateful to the anonymous reviewers for
their time and constructive suggestions.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.061770/-/DC1
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